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Analysis of the RNA binding specificity of the human tap protein, a constitutive transport element-specific nuclear
1Department of Genetics, Duke University Medical Center, Durham, North Carolina 27710, USA.
Abstract:
The human Tap protein has been proposed to mediate Mason Pfizer monkey virus constitutive transport element (CTE)-dependent nuclear RNA export and may also play a role in global mRNA export. Here, we have used in vivo assays, in both yeast and human cells, together with in vitro assays, to further characterize the RNA binding properties of Tap, which has been proposed to contain a novel leucine-rich RNA binding motif. Using the yeast three hybrid assay, we selected RNA molecules that retain Tap binding activity from a pool of randomized CTE sequences. The recovered RNA sequences differed only minimally from the wild-type CTE yet all displayed lower affinity for Tap both in vivo and in vitro. Analysis of the RNA export activity of the recovered CTE variants revealed that Tap affinity was highly predictive of CTE biological activity. Together, these observations provide additional evidence supporting the identification of Tap as the direct cofactor for CTE function and demonstrate that RNA binding by Tap is highly sequence specific.
Insights
The human Tap protein directly binds RNA in a sequence-specific manner, crucial for Mason Pfizer monkey virus RNA export. This specificity is key to Tap protein
Area of Science:
- Molecular Biology
- Virology
- Cell Biology
Background:
- The human Tap protein is implicated in nuclear RNA export, particularly for Mason Pfizer monkey virus constitutive transport element (CTE).
- Tap protein is hypothesized to possess a unique leucine-rich RNA binding motif and may influence global mRNA export.
Purpose of the Study:
- To further characterize the RNA binding properties of the human Tap protein.
- To investigate the sequence specificity of Tap protein's RNA binding and its role in CTE-dependent RNA export.
Main Methods:
- Utilized in vivo assays in yeast and human cells.
- Employed in vitro binding assays.
- Used the yeast three-hybrid system to select RNA sequences with Tap binding activity from randomized CTE pools.
Main Results:
- Selected RNA sequences showed minimal deviation from wild-type CTE but exhibited reduced affinity for Tap protein.
- Tap protein binding affinity strongly correlated with the biological activity of CTE variants in RNA export.
- Demonstrated that Tap protein's RNA binding is highly sequence-specific.
Conclusions:
- Provides strong evidence that Tap protein is the direct cofactor mediating CTE function.
- Confirms the sequence-specific nature of Tap protein's interaction with RNA.
- Highlights the importance of specific RNA sequences for Tap-mediated nuclear export.